The transplantation or transfusion of therapeutic cells has been pursued as very actively by researchers over the last decade, and, for progenitor and stem cell therapy, remarkable progress has been made in animal disease models. There are several imaging modalities that are capable of non-invasively and repetitively imaging targeted cells and cellular processes in living organisms. Such imaging modalities include γ-camera imaging, single photon emission computed tomography (SPECT), and positron emission tomography or PET (which use radioactive labels), bioluminescence imaging, and MR imaging. When these modalities are compared, however, only MR imaging offers near-cellular spatial resolution, with the potential of imaging only a few cells.
MR imaging, γ-camera imaging, and SPECT do require tagging of the cells with a suitable marker, while reporter genes have been developed for bioluminescent and PET imaging (although PET can also use tagged cells1). As for PET imaging, studies by Gelovani aka Tjuvajev and others have shown that HSV-tk can be used [Tjuvajev, J. G. et al. Noninvasive imaging of herpes virus thymidine kinase gene transfer and expression: a potential method for monitoring clinical gene therapy. Cancer Res 56, 4087-4095 (1996); Tjuvajev, J. G. et al. Imaging the expression of transfected genes in vivo. Cancer Res 55, 6126-6132 (1995); and Gambhir, S. S. et al. Imaging adenoviral-directed reporter gene expression in living animals with positron emission tomography. Proc Natl Acad Sci USA 96, 2333-2338 (1999)]. The dopamine type 2 receptor, the somatostatin receptor type 2, and the bombesin receptor systems have also been used as potential nuclear imaging probes of gene expression, although to lesser extents [Gambhir, S. S. et al. Imaging adenoviral-directed reporter gene expression in living animals with positron emission tomography. Proc Natl Acad Sci USA 96, 2333-2338 (1999) and Rogers, B. E. et al. Localization of iodine-125-mIP-Des-Met14-bombesin (7-13)NH2 in ovarian carcinoma induced to express the gastrin releasing peptide receptor by adenoviral vector-mediated gene transfer. J Nucl Med 38, 1221-1229 (1997)]. In the context of cellular imaging, the HSV1-tk reporter system has been used to image intracellular pathways, such as the induction of p53 expression and T-cell activation [Ponomarev, V. et al. Imaging TCR-dependent NFAT-mediated T-cell activation with positron emission tomography in vivo. Neoplasia 3, 480-488 (2001)], and is currently being explored for imaging of stem cell [Ivanova, A. et al. Imaging adoptive stem cell therapy with HSV-tk/GFP reporter gene. Mol Imaging 1, 208-209 (2002)] and T cell trafficking [Koehn, G. et al. Serial in vivo imaging of the targeted migration of human HSV-TK-transduced antigen-specific lymphocytes. Nat Biotechnol 21, 405-413 (2003) and Dubey, P. et al. Quantitative imaging of the T cell antitumor response by positron-emission tomography. Proc Natl Acad Sci USA 100, 1232-1237 (2003)].
For bioluminescent imaging, firefly luciferase is commonly used, but other enzymes, i.e., Renilla luciferase, have been developed as well [Bhaumik, S. & Gambhir, S. S. Optical imaging of Renilla luciferase reporter gene expression in living mice. Proc Nail Acad Sci USA 99, 377-382 (2002)]. The reporter genes have been successfully used for the detection of homing sites of prokaryotes [Hardy, J. et al. Extracellular replication of Listeria monocytogenes in the murine gall bladder. Science 303, 851-853 (2004)] and viruses [Luker, G. D. et al. Noninvasive bioluminescence imaging of herpes simplex virus type 1 infection and therapy in living mice. J Virol 76, 12149-12161 (2002)]. As for imaging of eukaryote cells, transfected islet cell grafts have been followed without adverse effects on islet function [Lu, Y. et al. Bioluminescent monitoring of islet graft survival after transplantation. Mol Ther 9, 428-435 (2004), and trafficking/migration of C17.2 cells has been successfully visualized in animal model of stroke and intracranial gliomas. Similarly, lymphocyte trafficking has been monitored serially over time.
The reporter gene strategy is so sensitive that even single hematopoietic bone marrow stem cells can be detected following transfusion and replication in the host (Contag, SMI 2003). Recently, cells have also been transfected with double or triple reporter genes, allowing the application of multiple imaging modalities. A hallmark for the use of these reporter genes in both PET and bioluminescent imaging is that they require the administration of a substrate, i.e., luciferin, coelenterazine, FIAU, or FHBG. The use of reporter genes has a narrow time window of imaging, unless repeated injections of substrate are applied, and some agents are unable to cross an intact blood-brain barrier. An example of an endogenous reporter gene that does not require substrate administration is green fluorescent protein (EGFP). However, so far, optical imaging cannot be used in larger animals such as mammals.
For the cells of interest to be visualized using MR imaging, they need to be magnetically labeled in order to be discriminated from the surrounding native tissue. For both applications, gadolinium chelates may be used, but these agents exhibit low relaxivities, which further decrease upon cellular internalization. Furthermore, gadolinium is not biocompatible, and very little is known about its potential toxicity following cellular dechelation over time. Because of their biocompatibility and strong effects on T2 and T2* relaxation, superparamagnetic iron oxides (SPIO) are now the preferred magnetic label for use in MR cell tracking. As they are composed of thousands of iron atoms, they defeat the inherent low contrast agent sensitivity of MRI. They also have other convenient properties, including the ability to be detected by light (Prussian Blue stain) and electron microscopy, and the ability to change their magnetic properties according to size, with the potential to reveal their structural (bound) conformation.
Despite these recent advances, there are several limitations that hamper exploiting the full potential of high-resolution MRI cell tracking using iron oxide particles. The amounts of iron necessary for sufficient detection are in the picogram range per cell. Few detailed studies on the potential adverse effect of iron on normal cellular function have been performed. As iron plays a role in many metabolic pathways, it would not be surprising to encounter negative effects in labeled cells that are not “professional scavengers or biodegraders” (i.e., not macrophages). Indeed, while Feridex labeling of human mesenchymal stem cells was found not to affect viability or proliferation, it has been found that the differentiation of mesenchymal stem cells (MSCs) into chondrocytes was markedly inhibited [Bulte, J. W. M., Kraitchman, D. L., Mackay, A. M. & Pittenger, M. F. Chondrogenic differentiation of mesenchymal stem cells is inhibited after magnetic labeling with ferumoxides. Blood 104 (2004), while adipogenic and osteogenic differentiation were not affected. The unexpected inhibition of the mesenchymal pathway into chondrogenic differentiation was mediated by the Feridex and not the PLL, through an as yet unknown mechanism. These results highlight the need for caution in the use of Feridex-labeling for certain cell tracking applications.
More generally, a potential limitation of SPIO-based cell tracking lies in the fact that any non-genetic material carried by the cell is eventually degraded, can exit the cell, or be incorporated into neighboring cells following cell death. It is thus unclear how long after labeling cells can be visualized and monitored reliably. Moreover, fast proliferating, dividing cells can rapidly dilute the iron label by cell division. As the effective concentration of these contrast materials is reduced with every cell division, the detection efficiency also is reduced. To date, all MR cell tracking approaches and genetically encoded MR reporters utilize the same contrast mechanism, (super)paramagnetic relaxation enhancement, allowing detection of only one type of labelled cell.
When reviewing the available MR literature, it is clear that the development of a suitable reporter gene for MR imaging has long been an elusive goal. Early attempts have used creatine kinase and cytosine deaminase [Koretsky, A. P., Brosnan, M. J., Chen, L. H., Chen, J. D. & Van Dyke, T. NMR detection of creatine kinase expressed in liver of transgenic mice: determination of free ADP levels. Proc Natl Acad Sci USA 87, 3112-3116 (1990) and Stegman, L. D. et al. Noninvasive quantitation of cytosine deaminase transgene expression in human tumor xenografts with in vivo magnetic resonance spectroscopy. Proc Natl Acad Sci USA 96, 9821-9826 (1999)] for spectroscopic imaging but, again, these encode for enzymes that convert substrates (adenine diphosphate and 5-fluorocytosine, respectively). Meade et al. have used the lacZ gene encoding for β-galactosidase and shown that embryonic cells injected with eGad can be selectively visualized [Louie, A. Y. et al. In vivo visualization of gene expression using magnetic resonance imaging. Nat Biotechnol 18, 321-325 (2000)]. LacZ-transfected cells have recently also been used to convert the NMR-sensitive molecule, 4-fluoro-2-nitrophenyl-β-D-galactopyranoside [Cui, W. et al. Novel NMR approach to assessing gene transfection: 4-fluoro-2-nitrophenyl-beta-D-galactopyranoside as a prototype reporter molecule for beta-galactosidase. Magn Reson Med 51, 616-620 (2004)]. Use of ferritin as an MR reporter gene has been recently reported [Cohen, B., Dafni, H., Meir, G., Harmelin, A. & Neeman, M. Ferritin as an endogenous MRI reporter for noninvasive imaging of gene expression in C6 glioma tumors. Neoplasia 7, 109-117 (2005); Genove, G., Demarco, U., Xu, H., Goins, W. F. & Ahrens, E. T. A new transgene reporter for in vivo magnetic resonance imaging. Nat Med (2005)]. Weissleder and Basilion et al. were able to detect transferrin-receptor-overexpressing tumors using a superparamagnetic transferrin probe [Weissleder, R. et al. In vivo magnetic resonance imaging of transgene expression. Nat Med 6, 351-355 (2000)].
Chemical exchange saturation transfer (CEST) MR imaging, which is shown schematically in FIG. 1, is a relatively new technique in which low-concentration marker molecules are labeled by saturating their exchangeable protons (e.g., hydroxyl, amine, amide, or imino protons) by radio-frequency (RF) irradiation. If such saturation can be achieved rapidly (i.e., before the proton exchanges), exchange of such labeled protons with water leads to progressive water saturation, allowing indirect detection of the solute via the water resonance through a decrease in signal intensity in MRI [Ward, K. M., Aletras, A. H. & Balaban, R. S. A new class of contrast agents for MRI based on proton chemical exchange dependent saturation transfer (CEST). J Magn Reson 143, 79-87 (2000)]. Each CEST contrast agent can have a different saturation frequency, which depends on the chemical shift of the exchangeable proton. The magnitude of proton transfer enhancement (PTE) due to this effect, and the resulting signal reduction from equilibrium (S0) to saturated (S), are given by [Goffeney, N., Bulte, J. W., Duyn, J., Bryant, L. H., Jr. & van Zijl, P. C. Sensitive NMR detection of cationic-polymer-based gene delivery systems using saturation transfer via proton exchange. J Am Chem Soc 123, 8628-8629 (2001)]:
                              PTE          =                                                    N                ⁢                                                                  ⁢                                  M                  w                                ⁢                α                ⁢                                                                  ⁢                                  k                  ex                                                                                                  (                                          1                      -                                              x                        CA                                                              )                                    ⁢                                      R                                          1                      ⁢                      wat                                                                      +                                                      x                    CA                                    ⁢                                      k                    ex                                                                        ·                          {                              1                -                                  ⅇ                                                            -                                              [                                                                                                            (                                                              1                                -                                                                  x                                  CA                                                                                            )                                                        ⁢                                                          R                                                              1                                ⁢                                wat                                                                                                              +                                                                                    x                              CA                                                        ⁢                                                          k                              ex                                                                                                      ]                                                              ⁢                                          t                      sat                                                                                  }                                      ,                                  ⁢        and                            [                  Eq          .                                          ⁢          1                ]                                          (                      1            -                                          S                sat                            /                              S                0                                              )                =                                            PTE              ·                              [                CA                ]                                                    2              ·                              [                                                      H                    2                                    ⁢                  O                                ]                                              .                                    [                  Eq          .                                          ⁢          2                ]            “CA” is the contrast agent containing multiple exchangeable protons, xCA its fractional exchangeable proton concentration, α the saturation efficiency, k the pseudo first-order rate constant, N the number of exchangeable protons per molecular weight unit, and Mw the molecular weight of the CA. The exponential term describes the effect of back exchange and water longitudinal relaxation (R1wat=1/T1wat) on the transfer during the RF saturation period (tsat). This effect will be bigger when protons exchange faster, but the catch is that saturation must occur faster as well, which increases the radio-frequency power needed. In addition, the resonance of the particular protons must be well separated from water in the proton NMR spectrum (so that it can be irradiated selectively as illustrated in FIG. 1), which requires a slow exchange on the NMR time scale. This condition means that the frequency difference of the exchangeable protons with water is much larger than the exchange rate (Δω>k). Thus, the CEST technology becomes more applicable at higher magnetic fields or when using paramagnetic shift agents [Zhang, S., Merritt, M., Woessner, D. E., Lenkinski, R. E. & Sherry, A. D. PARACEST agents: modulating MRI contrast via water proton exchange. Acc Chem Res 36, 783-790 (2003)]. Any molecule that exhibits a significant PTE effect can be classified as a CEST (contrast) agent. The concept of these agents as MR contrast agents is somewhat similar to the chemical amplification of colorimetric labels in in situ gene expression assays. CEST agents can be detected by monitoring the water intensity as a function of the saturation frequency, leading to a so-called z-spectrum. In such spectra, the saturation effect of the contrast agent on the water resonance is displayed as a function of irradiation frequency. The water signal resonates at 4.7 ppm, which therefore shows complete direct saturation. When a sample (or tissue) contains exchangeable protons that have a separate MRI frequency (e.g., amide backbone protons at 8.3 ppm in peptides), their irradiation reduces the water signal.
CEST agents with two types of exchangeable protons that have different chemical shifts can be used to monitor pH based on ratiometric methods. In addition, Sherry et al. and Aime et al. have developed a new class of CEST agents containing a paramagnetic center, so-called PARACEST agents. In this type of agent, a lanthanide ion with a chelator binds water weakly, which can then exchange with bulk water. The bound water has a greater chemical shift than bulk water due to the paramagnetic lanthanide, and can “hop” between free and bound at a fast rate. In addition, Sherry et al. have characterized a series of complexes that have a wide range of exchange rates suitable for large contrasts. PARACEST agents, however, provide contrast only at millimolar concentrations.
In order to effectively develop cell-based therapies that will be applicable in the clinic, noninvasive cellular imaging techniques are required. These imaging techniques are needed to provide detailed information on the biokinetics of administered cells (either transplanted or transfused), and cell-tissue interactions, including preferred pathways of migration, and cell survival. In addition, within the hematological and immunological communities. There also is now increasing interest in obtaining a deeper understanding of the spatiotemporal dynamics of cell “homing” following intravenous injection of hematopoietic and white blood cells.
The references discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention.
It thus would be desirable to provide a new reporter gene that can be detected by CEST MR imaging with high sensitivity, and that does not require the administration of exogenous probes or substrates. It also would be particularly desirable to provide methods for making such a reporter gene as well as CEST based MRI methods the embody the use of such reporter genes.